JP2010539941A - Production of amino acids from sucrose in Corynebacterium luglutamicum - Google Patents

Abstract

Using sucrose as a carbon source, C.I. Methods and compositions for increasing the production of amino acids from glutamicum are described. In one embodiment, C.I. Increased production of L-lysine from glutamicum results in strains with mutations in the ptsF gene encoding the fructose-PTS enzyme that attenuate or block the transfer of fructose into the cell This is achieved using such strains if the growth is increased and production is increased by providing glucose isomerase in the fermentation medium. Glucose isomerase can be added or expressed exogenously in the strain and excreted in the medium. In certain embodiments, the medium also contains invertase.

Description

This application claims priority to pending US Provisional Patent Application No. 60 / 955,348, filed Sep. 26, 2007. The above application is incorporated by reference as if it were fully described herein again.

FIELD OF THE INVENTION The present invention relates generally to methods and compositions for producing amino acids by bacteria of the genus Corynebacterium or Brevibacterium using sucrose as a carbon source. More particularly, the present invention relates to the production of L-lysine from Corynebacterium glutamicum using sucrose as a carbon source.

Related Art Many commercial fermentations of Corynebacterium and Brevibacterium use glucose as a carbon source. Thus, many bacterial production cultures have been designed to optimize production rate and / or yield using glucose as a carbon source. Production of L-lysine, a commercially important amino acid, is a specific target for optimization.

  Due to costs and other possible considerations, the use of alternative non-glucose carbon sources may be preferred in some parts of the world. One possible non-glucose carbon source is sucrose. Sucrose can be obtained from, for example, sugar cane or sugar beet. Unfortunately, the transport and utilization of sucrose by microorganisms is often different from glucose, so the production of desirable amino acids or fine chemical products from many microorganisms using sucrose as a carbon source can cause reduced efficiency. There is. This may be especially the case when microorganisms that use sucrose as a carbon source are designed to grow optimally on glucose. This is also the case with Corynebacterium glutamicum, one of the most commonly used microorganisms for the production of amino acids such as L-lysine by fermentation.

  C. A metabolic pathway of sucrose utilization in glutamicum was proposed by Non-Patent Document 1. Wittmann et al., Corynebacterium glutamicum has a sucrose uptake mechanism that occurs by the phosphotransferase system (PTS), in which the glucose ring of sucrose is phosphorylated and subsequently hydrolyzed to glucose-6-phosphate and fructose in the cell. It was assumed that C. PTS in glutamicum is a membrane-bound EII protein complex named fructose-pts, sucrose-pts and glucose-pts, encoded by the ptsF, ptsS and ptsG genes, respectively, preferentially transporting fructose, sucrose and glucose, respectively. In a common carbohydrate transport system that utilizes a combination of two commonly shared cytoplasmic proteins, designated Enzymes I and Hpr, encoded by the ptsI and ptsH genes, respectively, that interact with a separate set of bodies (Non-patent document 2. There are also two pts genes named HCg12933 and NCg12934 encoding proteins with unknown specificity (ibid.).

  Wittmann et al. Further, the fructose resulting from hydrolysis is secreted from the cell and then re-imported through the fructose-PTS uptake system and the mannose PTS uptake system (the latter is now considered the same as glucose-PTS). Assumed. The presence of multiple sucrose uptake systems (sucrose-PTS, fructose-PTS, and glucose-PTS) and thus the presence of multiple carbon entry points into the cell may be an undesirable productivity of sucrose. Assumed as the reason.

  During lysine production on glucose, about 65% of the carbon goes to the pentose phosphate pathway (PPP) to produce NADPH used for lysine synthesis. However, when synthesizing lysine on sucrose, it is thought that a very low proportion of carbon goes to PPP because almost half of the total carbon enters the glycolysis as fructose-1,6-phosphate. .

  As illustrated in FIG. When glutamicum grows on sucrose, C.I. About 90% of the fructose that will enter glutamicum cells is believed to enter as fructose-1-phosphate through fructose-PTS. Fructose-1-phosphate is phosphorylated to produce fructose-1,6-phosphate. Mainly, 6-phosphofructokinase is mainly an irreversible enzyme, and C. cerevisiae grown on sucrose. It is believed that fructose-1,6-phosphate does not pass through the PPP pathway because glutamicum has very little fructose-1,6-biphosphatase activity. Thus, fructose-1,6-diphosphate preferentially enters the glycolysis and TCA cycle and does not provide reducing power for commercial level lysine synthesis.

  C. About 10% of the fructose entering glutamicum is believed to be taken up as fructose-6-phosphate by the glucose-PTS system. Fructose-6-phosphate increases the amount of carbon directed to the PPP flux by the action of glucose-6-phosphate isomerase, which functions in the gluconeogenesis direction to produce glucose-6-phosphate from fructose-6-phosphate. May contribute. One proposed metabolic pathway for sucrose utilization in Corynebacterium, including pathways through glycolysis and PPP shunts, is shown in FIG.

  Increased lysine production from Corynebacterium on sucrose has been evaluated. One method that can be used to increase production was described by [3].

  Georgi et al.'S strategy is to use a constitutive promoter to overexpress the fructose-1,6-bisphosphatase gene fbp. However, this strategy is optimized for growth on sucrose, but may lead to the creation of a strain of Corynebacterium having properties that may lead to suboptimal growth in glucose, which may be undesirable There is sex. This could potentially reduce the flexibility of the strain's economically viable application and use sucrose or glucose as a carbon source, depending on what is economically advantageous as conditions change It may not work as you can.

  Another contemplated strategy for increasing lysine production from Corynebacterium on sucrose is reported in US Pat. In this strategy, fructose-1,6-bisphosphatase is overexpressed. This is said to allow fructose-1,6-P to return to PPP and ultimately increase the amount of NADPH. The basic strategy of Georgi et al. And Becker et al. Is illustrated in FIG.

  Another proposal to possibly increase lysine production from Corynebacterium using sucrose as a carbon source was reported in [5]. Moon is a C.I. Culture medium from transformed strains during growth on sucrose, expression of Clostridium acetobutylic fructose kinase gene in glutamicum is otherwise exported during log phase growth of the parent strain lacking fructokinase activity It has been demonstrated to reduce the fructose discharged into it. Moon et al. Utilize fructose excreted better than mutants lacking fructose-pts activity, but ptsF mutants that express the fructokinase enzyme grow to higher optical densities and lack fructokinase activity. It has also been demonstrated that it can. C. Expression of fructokinase in glutamicum proceeds to a PPP shunt, possibly increasing lysine production, instead of excreting fructose from the cell and then re-importing it primarily as fructose 1-phosphate via the PTS system. It was hypothesized that it would be possible to convert to fructose-6-P. The diagram assumed by Moon et al. Is illustrated in FIG.

  The kinetics of lysine production using fructose and sucrose as carbon sources are reported in Non-Patent Document 6 and Non-Patent Document 7. E. to modified Corynebacterium cells. The introduction of the E. coli xylose isomerase gene has been reported in Non-Patent Document 8.

International Publication No. 2005/059139 Pamphlet

Wittmann et al., “Metabolic Fluxes in Corynebacterium glutamicum dying Lysine Production with Sucrose as Carbon Source”, Appl. & Enviro. Microbiol. (2004) 70 (12): 7277-7287 Tanaka et al., Microbiology (2008) 154, 264-274) Georgi et al., “Lysine and Glutamate Production by Corynebacterium glutamicum on Glucose, Fructose, and Sucrose: Roles of Malic Enzyme and Fructo 6 (2005) 7: 291-301 Becker et al., "Amplified Expression of Fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose of phosphates pathway and of lysine production on different carbon sources", Appl. Envir. Microbiol. (2005) 71: 8587-8596 Moon et al., “Analyses of enzyme II gene mutants for sugar transport and heterologous expression of frutokinase gene in Corynebactrum um. Lett. (2005) 244: 259-266 Kiefer et al., “Influence of glucose, fructose and sucrose as carbon sources on kinetics of stiometry of lysine production by Chum. Indus. Microbiol. & Biotech. (2002) 28: 338-343 Kiefer et al., “Comparative Metabolic Flex Analysis of Lysine-Producting Corynebacterium glutamicum Cultured on Glucose or Fructose”, Appl. & Envir. Microbiol. (2004) 70 (1): 229-239 Kawaguchi et al., “Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum”, Appl. & Envir. Microbiol. (2006) 72 (5): 3418-3428

  Although optimized for growth on sucrose, it would be desirable to produce a strain of a microorganism that retains favorable properties for growth on glucose. Where sucrose is available, such strains can result in a more economically preferred fermentative production of amino acids from sucrose, and such strains when sucrose is not available or not as rich or cheap as glucose Should be able to be used efficiently for the fermentative production of amino acids from glucose. The object of the present invention is to produce such strains. It is a further object of the present invention to produce lysine using strains made according to embodiments of the present invention. Preferably, the amount and / or rate of lysine production will be greater in bacteria produced in embodiments of the present invention. Of course, the invention as defined by the claims is not limited by its ability to meet one or more of the objects of the invention.

  The present invention relates to the production of amino acids by bacteria of the genus Corynebacterium or Brevibacterium using sucrose as a carbon source. In one aspect, the present invention provides novel strains of microorganisms that have attenuated or blocked the fructose transport mechanism and methods for their production. In another embodiment, for optimal growth on sucrose, C.I. A fermentation method using an enzyme in a medium is provided that takes advantage of the native characteristics of glutamicum endogenous PTS.

  In one embodiment, the microorganism can be fermented in a fermentation broth supplemented with glucose isomerase and / or invertase. In another embodiment, glucose isomerase and / or invertase can be expressed in cells and excreted into the medium. In another embodiment, the microorganisms of the invention have been mutated to have attenuated or blocked fructose transport or fructose excretion mechanisms. In yet another aspect, the microorganism can be engineered to express glucose isomerase and glucokinase in the cytoplasm to drive fructose imported via formation of glucose-6-phosphate in the direction of the PPP shunt. . A method for producing amino acids using these microorganisms, culture media, or both is also included in the embodiments of the present invention.

It is a figure which shows the wild type metabolism of sucrose and fructose in Corynebacterium. Moon et al., “Analyses of enzyme II gene mutants for sugar transport and heterologous expression of frutokinase gene in Corynebactrum um. Lett. 244: 259-266 (2005) shows the sucrose and fructose metabolic pathways of Corynebacterium according to embodiments of the present invention providing fructose-PTS knockout mutants, such as those described by. The Moon et al. PTS knockout mutant form is used in the present invention with the addition or secretion of glucose isomerase in the fermentation medium. FIG. 5 shows the sucrose and fructose metabolic pathways of Corynebacterium according to embodiments of the present invention in which invertase and glucose isomerase are added or secreted into the fermentation medium along with fructose-PTS knockout mutants, such as those described by Moon et al. is there. FIG. 5 shows the Corynebacterium sucrose and fructose metabolic pathways in which glucose isomerase has been cloned and expressed in mutant cells with both fructose-PTS knockout and fructose efflux transporter knockout. FIG. 2 shows a prior art sucrose and fructose metabolic pathway in which fructose-1,6-diphosphate is overexpressed in Corynebacterium. This pathway, WO2005 / 059139A2 and Becker et al., "Amplified Expression OF Fructose 1,6-bisphosphatase in C.glutamicum increases in vivo flux through the pentose of phosphates pathway and of lysine production on different carbon sources", Appl. Envir. Microbiol. 71: 8587-8596 (2005). FIG. 5 shows the prior art Corynebacterium sucrose and fructose metabolic pathways by Moon et al., Which also express fructose kinase.

  The foregoing background and summary of the invention and the following detailed description can assist those of ordinary skill in the art to better understand the present invention and / or facilitate the understanding of the present invention as described herein. The composition, bacterial strain and C.I. Included are citations of various references that provide an explanation of how to make glutamicum variants and genetically engineered expression vectors. Thus, rather than reproducing a prominent description of a number of references known in the art, all references cited herein are intended for such references to produce lysine and other amino acids. , Strain efficacy, methods of making variants and recombinants, strain characteristics and availability and C.I. To the extent that it teaches the fermentation process of glutamicum, it is hereby incorporated by reference. To the extent that any teaching in the incorporated reference contradicts the description provided herein, this description dominates.

  The present invention relates to the production of fine chemicals by bacteria of the genus Corynebacterium or Brevibacterium using sucrose as a carbon source. These fine chemicals may be, for example, amino acids or vitamins. Amino acids that can be produced include, but are not limited to, for example, L-lysine, L-tryptophan, L-methionine, L-threonine, and homoserine.

  It has been discovered in the present invention that an increase in NADPH levels in bacterial cells increases production yield, particularly in anabolic processes where NADPH is a limiting factor. Methods for transporting chemical energy from catabolism to reactions that require biosynthetic energy, such as the formation of amino acids, are in the form of hydrogen atoms or electrons.

In order to be effective as a reducing agent, a hydrogen atom must have considerable free energy. Such high energy hydrogen atoms are removed by dehydrogenases that catalyze the removal of hydrogen atoms from fuel molecules and their transport to specific coenzymes, in particular to the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP ⁺ ). Obtained from cellular fuel. This form of coenzyme, named NADPH, which is reduced or carries hydrogen, is a carrier of high energy electrons from catabolism to biosynthetic reactions that require electrons.

  Preferably, NADPH effectiveness is increased by increasing carbon flux through the oxidative branch of the pentose phosphate pathway. Theoretically, when glucose is metabolized exclusively in the pentose phosphate pathway, 12 molecules of NADPH are generated per glucose molecule, but are metabolized in the TCA cycle (also called tricarboxylic acid or citrate cycle). Only 2 molecules of NADPH are generated per molecule. Ishino, S et al., J. MoI. Gen. Appl. Microbiol. 37: 157-165 (1991). The present invention provides a method for producing L-amino acids by culturing modified bacterial cells with increased carbon flux of sucrose and sucrose products through the pentose phosphate pathway.

  The pentose phosphate pathway (“PPP”), also referred to as the hexose phosphate pathway, is an alternative pathway for glucose catabolism. The pentose phosphate pathway produces NADPH and is more active under lysine fermentation conditions. Ishino, S et al., J. MoI. Gen. Appl. Microbiol. 37: 157-165 (1991). Since sucrose is naturally sent through PPP, there is relatively little NADPH formed from sucrose. PPP is further discussed in US Pat. No. 6,830,903 to O'Donohue et al.

  In one aspect, the present invention provides one or more novel microbial strains with increased NADPH levels as a result of attenuated or blocked fructose uptake mechanisms. Such attenuation and blocking can be achieved, for example, by disruption or removal of the gene encoding the enzyme in the fructose-PTS uptake pathway. Such an enzyme can be, for example, fructose-PTS enzyme II (SEQ ID NO: 1) encoded by the ptsF gene (SEQ ID NO: 2). An example of the destruction of ptsF is described in Example 1 below. One skilled in the art can recognize that other genes can also be effectively disrupted or attenuated to reduce or eliminate fructose uptake.

  The fructose derived from sucrose uptake is microorganisms grown on sucrose, for example by using a fructose-PTS enzyme variant, as described by Moon et al. The reduction or elimination of fructose uptake in would necessarily lead to fructose accumulation in the medium. This fructose can be converted to glucose by the action of glucose isomerase. This glucose occurs when fructose that otherwise enters the cell preferentially via the fructose-PTS enzyme is first converted to fructose-1-phosphate, etc., and then the glucose phosphotransferase system (glucose- Rather than being preferentially taken up by PTS) and proceeding preferentially through the glycolysis, it will be immediately converted to glucose-6-phosphate that enters the PPP shunt. Since glucose is transported into cells by glucose-PTS much more efficiently than fructose, the isomerization equilibrium will be driven towards converting excreted fructose into glucose. A proposed metabolic pathway using sucrose in this embodiment of the present invention is shown in FIG.

  In this aspect of the invention, glucose isomerase is included in the medium. Inclusion of glucose isomerase in the medium is accomplished, for example, through adding the enzyme before the start of fermentation, through adding the enzyme during fermentation, or through continuous addition of enzyme as needed. be able to. Glucose isomerase can be added as a liquid enzyme and an immobilized enzyme, or a mixture of the two. In a preferred embodiment of the invention, sufficient glucose isomerase is added to maintain a balance of fructose to glucose conversion. Glucose isomerase is commonly used in the commercial production of high fructose corn syrup from dextrose and is readily available in industrial quantities from commercial manufacturers such as Genecor (Rochester, NY), a division of Danisco US. Is possible.

  In another embodiment of this aspect, the microorganism can include a gene that is transcribed to produce glucose isomerase that is excreted into the medium. This can replace or supplement the addition of glucose isomerase to the medium as described above. For example, the gene for glucose isomerase (SEQ ID NO: 3) (EC 5.3.1.5 D-xylose ketol-isomerase) has been cloned from organisms such as Streptomyces rubiginosus, and C.I. It can be expressed in strains of glutamicum. One example of glucose isomerase is shown in SEQ ID NO: 4. Genes encoding glucose isomerase can also be used in other organisms, for example, the gene can be obtained from a strain of Brevibacterium. See, for example, Tateno et al., Applied Microbiology and Biotechnology, (2007) 77: 533-541 and Salim et al., Appl. Environ. Microbiol. (1997) 63: 4392-4400, for example, C.I. a vector containing the signal sequence necessary to produce the fusion protein to be excreted into the medium, such as the vector and signal sequence of the PS2 protein of the csoB gene of glutamicum. Vectors containing promoters for expressing recombinant genes in glutamicum are well known in the art.

  In another embodiment, the present invention provides a C.I. having a knockout mutation in a fructose-PTS enzyme as described above. In addition to glucose isomerase in the growth of glutamicum, the addition of invertase enzyme to the medium is included. The proposed metabolic pathway of this embodiment is shown in FIG. The addition of invertase will form glucose and fructose directly from at least a portion of the sucrose carbon feed, further increasing the amount of extracellular fructose that can be used for conversion to glucose by glucose isomerase. This embodiment avoids the production of fructose-1-phosphate by fructose-PTS and at the same time uses both glucose-PTS enzyme and sucrose-PTS enzyme to keep driving glucose-phosphate production in cells. Make full use of. Commercial quantities of invertase are readily available from manufacturers such as Novozymes North America, (Franklinton, NC).

  In yet another aspect, the present invention relates to fructose-PTS enzyme and C. aeruginosa grown on sucrose. It provides a double mutation in both the fructose efflux transporter involved in the elimination of fructose from glutamicum cells, thus eliminating or reducing the activity of the efflux transporter. Embodiments of this aspect can further include an increase in intracellular expression of glucose isomerase and glucokinase such that fructose retained in the cell is rapidly converted to glucose and then glucose-6-phosphate. . One example of glucokinase is glucose kinase according to SEQ ID NO: 5. C. One glk gene (SEQ ID NO: 6) encoding glucose kinase in glutamicum is described in Park et al., “Characterization of glk, a gene coding for glucose kinase of C. glutamicum”, FEMS Microbiol. Lett. 188: 209-215 (2000). Embodiments that overexpress glucokinase are sufficient for the strain to convert excess glucose formed by glucose isomerase from retained fructose for final conversion to glucose-6-phosphate. It is particularly beneficial if it does not otherwise have any endogenous glucokinase activity. The metabolic pathway for sucrose utilization in this embodiment is shown in FIG.

  The uniqueness of the fructose efflux transporter is not known, but its presence is reported in C.I. Recognized by the observed transport of fructose that occurs when glutamicum cells are grown using sucrose as a carbon source. Thus, when starting with a strain having a fructose PTS mutation, such as the ptsF mutant described by Moon et al., The strain is subject to normal random mutagenesis by chemical means and otherwise on sucrose. Cells that can grow in but do not excrete fructose are selected. One method for selecting cells that do not transport fructose is described by Dominguez et al., “New and simple plate test for screening transactivity activity of fungi,” Volume I, 19:23, Rev. Iberoom Mr. . Dominguez et al. Teach an assay in which cells with transfructosylating activity can be identified by blue light around cell colonies grown on agar. Therefore, C.I. glutamicum ptsF mutants will produce such blue light when grown on sucrose minimal medium, whereas mutants lacking fructose efflux transporter activity will not have circular light and thus The selection of the mutant is C.I. It is only a matter of visual screening of chemically mutagenized populations of glutamicum. Furthermore, those skilled in the art will recognize multiple assays that can be used to detect fructose and / or glucose confluence in the fermentation medium. For example, high pressure liquid chromatography (HPLC) can be used, or conventional colorimetric assays can be used, for example, with kits supplied from R-Biopharm AG.

  The following examples are provided as general guidance for practicing various aspects of the invention and are not intended to be limiting.

Example 1
Example 1 discloses the disruption of fructose-PTS enzyme II (SEQ ID NO: 1) encoded by the ptsF gene (SEQ ID NO: 2). This results in an increase in fructose concentration in the growth medium of cultures growing on sucrose. This process results in a ptsF variant similar to that described by Moon et al.

  Using the following primers, C.I. Amplifies the 690 base pair internal region of the glutamicum ptsF gene. The primers are as follows.

ＰＣＲ増幅条件を、以下のように使用した。ＰＣＲ反応の最終的な体積は、１００μｌである。５０ｎｇの高分子量のＤＮＡおよび２．５単位のＴａｑポリメラーゼとともに、１００ｎｇの各プライマーを使用する。反応緩衝液を製造業者が推奨する濃度で入れ、ｄＮＴＰも２００μＭの最終濃度で入れる。以下のようなサイクリングパラメーターを使用する：９４℃で１分間、続いて９４℃で３０秒、５８℃で３０秒、７２℃で１分間（３０サイクル）、７２℃で７分間、続いて４℃。ＰＣＲ断片をＨｉｎｄＩＩＩで消化し、カナマイシン耐性遺伝子を有する自殺ベクターｐＢＧＳ１３１中へクローン化し、結果として生じるクローンを使用して、Ｃ．ｇｌｕｔａｍｉｃｕｍを形質転換する（ＮＲＲＬ １１４７４）。組み込み体を、１０μｇ／ｍｌのカナマイシンを含有する培地プレート上で選択する。フルクトース−ＰＴＳ系のノックアウトを、細胞をスクロース上で増殖した場合の培地中のフルクトースの蓄積によって確認する。

PCR amplification conditions were used as follows. The final volume of the PCR reaction is 100 μl. 100 ng of each primer is used with 50 ng of high molecular weight DNA and 2.5 units of Taq polymerase. The reaction buffer is added at the concentration recommended by the manufacturer, and dNTP is also added at a final concentration of 200 μM. The following cycling parameters are used: 94 ° C. for 1 minute, followed by 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, 72 ° C. for 1 minute (30 cycles), 72 ° C. for 7 minutes, followed by 4 ° C. . The PCR fragment was digested with HindIII and cloned into the suicide vector pBGS131 carrying the kanamycin resistance gene and the resulting clone was used to transforming glutamicum (NRRL 11474). The integrants are selected on media plates containing 10 μg / ml kanamycin. Knockout of the fructose-PTS system is confirmed by the accumulation of fructose in the medium when the cells are grown on sucrose.

(Example 2)
Example 2 illustrates the addition of glucose isomerase to a lysine fermentation medium containing sucrose.

  An L-lysine producing strain of Corynebacterium, eg, strain deposited as NRRL B-11474, is grown in 20 ml of seed medium at about 30 ° C. for about 18 hours. 2 ml of this system is then transferred to a fermentation medium containing sucrose as at least one component of the carbon source and grown at 30 ° C. for 24 hours. Glucose isomerase is added to the fermentation medium as either a liquid enzyme or an immobilized enzyme. Enough enzyme is first added to maintain equilibrium, with about half of the sugar being fructose and the other half being glucose. As will be appreciated by those skilled in the art, the exact amount of glucose isomerase depends on the fermentation conditions. Continuous addition of glucose isomerase may be necessary to keep the activity of glucose isomerase high enough for the conversion of fructose to glucose.

(Example 3)
Example 3 is a C.I. The production of fructose excretion mutants in a background strain of glutamicum ptsF mutant is described.

C. The glutamicum ptsF mutant can be used to create a putative fructose efflux transporter mutant. C. We will mutate the glutamicum ptsF mutant. The ptsF mutant was grown to mid-log phase, pelleted by centrifugation, 2 ml of sterile TM buffer (Tris-HCl 6 g / l, 5.8 g / l maleic acid, adjusted to pH 6.0 with KOH, (NH4) 2SO4 1.0 g / l, Ca (NO3) 2 5 mg / l, MgSO4.7H2O 0.1 g / l, FeSO4.7H2O 0.25 mg / l). To 2 ml of cell suspension, add 50 μl of a 5.0 mg / l solution of N1-nitro-N-nitrosoguanidine (NTG) and then incubate at 30 ° C. for 30 minutes. 10 ml of TM buffer was then added, the cells were pelleted by centrifugation, washed twice in TM buffer, and 4.0 ml of 0.1 M NaH2PO4 (pH 7.2 adjusted using KOH). Resuspended in phosphate buffer). The cell suspension was further diluted to achieve about 200-300 colonies per plate and plated out on the following minimal medium.
(NH4) SO4 10 g / l
KH2PO4 1g / l
MgSO4 ^* 7H2O 0.4 g / l
NaCl 1g / l
Urea 2.5g / l
MnSO4 ^* H2O 0.01 g / l
FeSO4 ^* 7H2O 0.01 g / l
L-alanine 0.5g / l
L-methionine 0.5 g / l
L-threonine 0.25 g / l
Biotin 0.05mg / l
Thiamine 0.2mg / l
Niacinamide 0.05g /
Sucrose 1g / l
Agar 15g / l.

  After colony growth (2-5 days) at 40 ° C. methylthiazolyldiphenyl-tetrazolium bromide (MTT) (0.2 mg / ml), phenazine methosulfate (2.5 mg / l), fructose dehydrogenase 2 U / ml Plates were covered with soft agar (0.7% w / v) and pH 5.0 phosphate citrate buffer. Colonies that secrete fructose have a blue circular light, and colonies that do not secrete have no circular light. By picking, purifying and testing colonies without blue light, they grow on sucrose minimal medium (same as above but without agar) and determine whether fructose is in the medium. Make sure that it does not secrete fructose. Those that do not secrete fructose but grow on sucrose lack the fructose efflux transporter.

Example 4
In Example 4, C.I. Figure 3 illustrates intracellular expression of glucokinase and glucose isomerase in a ptsF mutant strain of glutamicum.

  Construction of a replicating plasmid containing glucokinase (SEQ ID NO: 6) and glucose isomerase (SEQ ID NO: 3). The following primers are used to amplify the gene for glucose isomerase from Streptomyces rubiginosus DNA by PCR. Forward primer,

は、遺伝子の発現に必要なｔａｃプロモーターおよびリボソーム結合部位を含有する。逆方向プライマーは、

Contains the tac promoter and ribosome binding site required for gene expression. The reverse primer is

である。これら２つのプライマーは、ｔａｃプロモーターに作動可能に連結しているグルコースイソメラーゼ遺伝子を増幅する。この断片を、ｐＤ１０のＳｍａＩ部位（米国特許第７，１４１，３８８号）中へ直接クローン化する。クローンを、正しい方向にある１つを求めてスクリーニングする。このプラスミドは、ｐＤ１０ｘｙｌＡと称される。

It is. These two primers amplify the glucose isomerase gene operably linked to the tac promoter. This fragment is cloned directly into the SmaI site of pD10 (US Pat. No. 7,141,388). Clones are screened for one in the correct orientation. This plasmid is called pD10xylA.

  Using the following primers, C.I. The glucokinase gene from glutamicum is cloned.

  The forward primer is

であり、
逆方向プライマーは、

And
The reverse primer is

である。

It is.

  These two primers amplify the glucokinase gene that will be cloned directly into the EcoRV site in pD10xylA. Again, clones with the glucokinase gene are screened for the correct orientation.

  The correct clone, called pD10xylAglk, contains the genes for glucose isomerase and glucokinase in a synthetic operon with expression controlled by the tac promoter. The plasmid pD10xylAglk is introduced by electroporation into the fructose efflux transporter variant described in Example 3, as described in US Pat. No. 7,141,388. Colonies are selected for resistance to chloramphenicol. These colonies contain the plasmid pD10xylAglk.

  Although the present invention has been described in some detail in detail above by way of illustration and example for the purpose of clarity of understanding, those skilled in the art will recognize the conditions, formulations and other parameters, along with a broad and equivalent range. It will be apparent with the advantages of the disclosure, that the invention may be practiced by modifying or changing the invention. Furthermore, it will be apparent to those skilled in the art, together with the advantages of the present disclosure, that such modifications or changes are intended to be included within the scope of the appended claims.

  All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each publication, patent or patent application is expressly incorporated by reference. And as if individually indicated, are incorporated herein by reference to the same extent. None of the publications, patents and patent applications mentioned in this specification are admitted to be prior art.

Claims (14)

A method of producing amino acids by fermentation,
C. sucrose as the carbon source and fructose-PTS enzyme II transferase activity is attenuated or blocked in the fermentation medium containing an amount of glucose isomerase effective to convert fructose to glucose in the fermentation medium. growing a glutamicum strain and fermenting the microorganism to produce the amino acid.   2. The method of claim 1 comprising the amino acid being L-lysine.   2. The method of claim 1, wherein the fructose-PTS attenuation or blockade is effected by disruption or deletion of the ptsF gene of the strain.   The method of claim 1, wherein the glucose isomerase is exogenously added to the fermentation medium.   The glucose isomerase is the C.I. 2. The method of claim 1, wherein the method is produced in a glutamicum strain and excreted from the strain into the medium.   The method of claim 1, wherein the fermentation medium further comprises an amount of invertase effective to convert a portion of the sucrose to fructose and glucose in the fermentation medium. A method of producing amino acids by fermentation,
In a fermentation medium containing sucrose as a carbon source, the transferase activity of fructose-PTS enzyme II is attenuated or blocked; growing the strain further comprising a mutation that attenuates or blocks fructose excretion from the glutamicum strain;
Fermenting the strain to produce the amino acid.   The method of claim 1, wherein glucose kinase is overexpressed in the strain.   The method of claim 1, wherein glucose isomerase is overexpressed in the strain.   2. The method of claim 1, wherein glucose kinase and glucose isomerase are overexpressed in the strain.   Fructose C.I. C. having a first mutation in the ptsF gene that attenuates or blocks transfer into a glutamicum strain and further has a mutation that attenuates or reduces the output of fructose from the strain. glutamicum strain.   C. above. The recombinant nucleic acid of claim 11 further comprising a recombinant nucleic acid encoding means configured to overexpress at least one of glucose isomerase and glucose kinase in a glutamicum strain. glutamicum strain.   C. When a glutamicum strain is grown on a medium containing sucrose, it has a first mutation in the ptsF gene that attenuates or blocks the transfer of fructose into the strain and expresses glucose isomerase; Having a recombinant nucleic acid encoding a means configured to be excreted from the strain into the fermentation medium; glutamicum strain.   C. C. having mutations that attenuate or reduce fructose excretion from Glutamicum strains. glutamicum strain.

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